The LRRK2 gene encodes a protein kinase and it has been disclosed that missense mutations in this gene can lead to a number of diseases such as various neurodegenerative diseases including Parkinson's disease. Additionally, LRRK2 is genetically linked to precancerous conditions and cancer; autoimmune disorders such as Crohn's disease, rheumatoid arthritis and psoriasis, and leprosy. For instance, it has been suggested that certain mutations in LRRK2 can lead to Parkinson's disease through the up-regulation of the kinase activity of the protein kinase. It is suspected that this protein kinase may be over-active in Parkinson's disease.
There has been much interest raised by the recent discovery that different autosomal dominant point mutations within the gene encoding for LRRK2 predispose humans to develop late-onset Parkinson's disease (OMIM accession number 609007), with a clinical appearance indistinguishable from idiopathic Parkinson's disease. See Paisan-Ruiz et al. (2004), “Cloning of the gene-containing mutations that cause PARK 8-linked Parkinson's disease.” Neuron. 44, 595-600; Mata et al. (2006), “LRRK2 in Parkinson's disease: protein domains and functional insights.” Trends Neurosci. 29, 286-293; Taylor et al. (2006), “LRRK2: a common pathway for Parkinsonism, pathogenesis and prevention?” Trends Mol. Med. 12, 76-82. The genetic analysis undertaken to date indicates that mutations in LRRK2 are relatively frequent, not only accounting for 5-10% of familial Parkinson's disease, but also being found in a significant proportion of sporadic Parkinson's disease cases. See Farrer et al. (2005), “LRRK2 mutations in Parkinson disease.” Neurology 65, 738-740; and Zabetian et al. (2005), “A clinic-based study of the LRRK2 gene in Parkinson disease yields new mutations.” Neurology 65, 741-744.
Little is known about how LRRK2 is regulated in cells, what its physiological substrates are, and how mutations cause or increase risk of Parkinson's disease. The domain structure of LRRK2 is depicted in WO 2011/141756 A1, the disclosure of which is incorporated herein by reference. See FIG. 1 therein, which also shows mutations which have been reported in patients with Parkinson's disease. The defining feature of the LRRK2 enzyme is a leucine rich repeat (LRR) motif (residues 1010-1291), a Ras-like small GTPase (residues 1336-1510), a region of high amino acid conservation that has been termed the C-terminal of Ras complex (COR) domain (residues 1511-1878), a protein kinase catalytic domain (residues 1879-2132) and a C-terminal VVD40 motif (2231-2276). See Bosgraaf et al. (2003), “Roc, a Ras/GTPase domain in complex proteins.” Biochim. Biophys. Acta. 1643, 5-10; and Marin (2006), “The Parkinson disease gene LRRK2: evolutionary and structural insights.” Mol. Biol. Evol. 23, 2423-2433.
The protein kinase domain of LRRK2 belongs to the tyrosine-like serine threonine protein kinases and is most similar to the kinase RIP (Receptor Interacting Protein), which play key roles in innate immunity signaling pathways. See Manning et al. (2002), “The protein kinase complement of the human genome.” Science 298, 1912-1934. Almost 40 single amino acid substitution mutations have been linked to autosomal-dominant Parkinson's disease. Mata et al., supra; Taylor et al., supra; WO 2011/141756 A1. It has also been reported that the most prevalent mutant form of LRRK2 accounting for approximately 6% of familial Parkinson's disease and 3% of sporadic Parkinson's disease cases in Europe, comprises an amino acid substitution of Gly2019 to a Ser residue. Gly2019 is located within the conserved DYG-Mg′-binding motif, in subdomain-VII of the kinase domain. Mata et al., supra. More recent reports suggest that this mutation enhances the autophosphorylation of LRRK2, as well as its ability to phosphorylate myelin basic protein 2-3-fold. West et al. (2005), “Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.” Proc. Natl. Acad. Sci. USA 102, 16842-16847; and Greggio et al. (2006), “Kinase activity is required for the toxic effects of mutant LRRK2ldardarin.” Neurobiol. Dis. 23, 329-341. These observations suggest that over-activation of LRRK2 predisposes humans to develop some forms of Parkinson's disease.
As discussed in “Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas”, Looyeng et al., Proceedings of the National Academy of Sciences of the United States of America (2011), 108(4), 1439-1444, S1439/1-S1439/10; language: English; database: CAPLUS, DOI:10.1073/pnas.1012500108; the receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). LRRK2 was identified and shown to be amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.
Missense mutations in LRRK2, as discussed above, cause late-onset Parkinson's disease (PD). In addition, common genetic variation in LRRK2 modifies susceptibility to Crohn's disease and leprosy. See “LRRK2 inhibition attenuates microglial inflammatory responses”, Moehle et al., Journal of Neuroscience (2012), 32(5), 1602-1611. Language: English, Database: CAPLUS, DOI:10.1523/JNEUROSCI.5601-11.2012.
Included among the genes identified as being associated with leprosy susceptibility or resistance, PARK2 and LRRK2 have been discussed as participating in the regulation of host-cell apoptosis. See “Leprosy susceptibility: genetic variations regulate innate and adaptive immunity, and disease outcome,” Cardoso et al., Future Microbiology (2011), 6(5), 533-549. Cardoso et al. further report that the same genes associated with leprosy are also associated with autoimmune (Crohn's disease, rheumatoid arthritis, psoriasis) or neurodegenerative diseases (Parkinson's and Alzheimer's).
Despite the discovery of the causal relationship between missense mutations within the gene encoding for LRRK2 and late-onset Parkinson's disease, the development of LRRK2 selective inhibitors has remained elusive. Thus, there remains a need for small molecule inhibitors of LRRK2.